Structural-Kinetic Analysis of Local Wild-Type and Mutants of Thermophile GH43 Bifunctional β-D-Xylosidase/α-L-Rrabinofuranosidase
Abstract
Xylan is a renewable component of hemicellulose, consisting of xylose linked by β‒1,4 glycosidic bonds. Complete xylan degradation requires various xylanolytic enzymes, including β‒xylosidase. GbtXyl43A, GH43 thermophilic bifunctional β-xylosidase/α-L-arabinofuranosidase derived from Geobacillus thermoleovorans IT-08, uses Glu-177 and Asp-14 as catalytic residues, with Asp-121 playing a pivotal role in catalysis. Mutations in Asp-121 reduced its stability and activity. Asp-121 mutation to glutamic acid (D121E), asparagine (D121N), or valine (D121V) diminished the structural stability of GbtXyl43A. The 3D structure of GbtXyl43A and its mutants at pH 6.0 showed a predominantly negative charge at Asp-121, indicating altered electrostatic charge distribution near the active site, affecting its catalytic function. Molecular docking simulations of GbtXyl43A and D121N, yielding binding energies of ‒7.2 kcal/mol and ‒6.7 kcal/mol. The kinetic parameters of GbtXyl43A and D121N were Vmax (3.35 × 10-3 and 0.10 × 10-3 mM/min), KM (2.84 and 4.56 mM), kcat (1.97 and 8.40 × 10-4 min-1), and kcat/KM (0.69 and 1.84 × 10-4 min-1mM-1). In-silico approach and analogous residue analysis indicated that Asp-121 functions as a pKa modulator essential for GbtXyl43A stability and catalytic activity. This study enhances the understanding of Asp-121 as the secondary aspartic acid residue that forms the catalytic triad of GH43 β-xylosidase.